It is our working hypothesis that mutations and altered expression of RB, p53, and PTEN, or those affecting regulatory genes involved in these pathways, produce a selective advantage for tumor growth and aggressive behavior in patients with soft tissue sarcomas (STS). This hypothesis will be tested by an integrated approach, analyzing human tumor samples and conducting genetic studies that include generation of in vitro and in vivo models. Aim 1. To define the clinical and biological implications of RB inactivation for proliferation and genomic stability in STS. We will characterize RB mutations, altered patterns of pRB and E2F1 expression, as well as upstream regulators (cyclin D, Cdk4). We will also assess Mad2 expression, an E2F1 transcriptional target that when deregulated causes chromosome instability and aneuploidy. We will assess the consequences of RB inactivation, identifying up- and down-regulated targets by high throughput studies. Aim 2. To characterize molecular alterations affecting the p53 pro-apoptotic response and define their clinical significance in STS. We will conduct a systematic characterization of TP53 mutations and p53 expression using a combination of methods. We will analyze regulatory events of the p53 pathway, focusing on Hdm2 and p14/Arf expression. We will study p53 apoptotic signaling, centering on Bax and Bcl-2, PUMA and NOXA, defining their impact on tumor progression and lack of response to particular treatments. The mechanistic consequences of p53 inactivation will be evaluated using functional assays. Aim 3. To determine the molecular mechanisms of tumor suppression by the Pten pathway in STS. We will establish the clinical relevance of detecting PTEN mutations and altered Pten expression. We will investigate the impact of downstream events, such as activation of Akt, mTOR and elF4E, as they participate in sarcomagenesis and STS progression. In addition, we are generating murine models to determine whether inactivation of Rb, p53 and Pten are required and cooperate for sarcoma development and progression. Our main objectives include: i) to assess the basis of the functional crosstalk between RB, p53, and PTEN pathways in tumor suppression;and ii) to translate basic and medical research findings into clinically applied studies. Our final translational goal is to produce a paradigm shift in clinical practice by incorporating biomarkers as robust decision tools to better guide the management of STS patients.